Discovery of potential natural therapeutics targeting cell wall biosynthesis in multidrug-resistant Enterococcus faecalis: a computational perspective.

Paclitaxel (PTX) is used to treat various cancers, but it also causes serious side effects and resistance. To better design similar compounds with less toxicity and more activity against drug-resistant tumors, it is important to clearly understand the PTX-binding pocket formed by the key residues of active sites on β-tubulin. Using a docking method, molecular dynamics (MD) simulation and density functional theory (DFT), we identified some residues (such as Arg278, Asp26, Asp226, Glu22, Glu27, His229, Arg369, Lys218, Ser277 and Thr276) on β-tubulin that are the active sites responsible for interaction with PTX. Another two residues, Leu371 and Gly279, also likely serve as active sites. Most of these sites contact with the "southern hemisphere" of PTX; only one key residue interacts with the "northern hemisphere" of PTX. These key residues can be divided into four groups, which serve as active compositions in the formation of an active pocket for PTX binding to β-tubulin. This active binding pocket enables a very strong interaction (the strength is predicted to be in the range of -327.8 to -365.7 kJ mol(-1)) between β-tubulin and PTX, with various orientated conformations. This strong interaction means that PTX possesses a high level of activity against cancer cells, a result that is in good agreement with the clinical mechanism of PTX. The described PTX pocket and key active residues will be applied to probe the mechanism of tumor cells resistant to PTX, and to design novel analogs with superior properties.

In-vitro release study of Pt(II) and Fe(III) metallocefotaxime drug candidates in pH dependent releasing mediums mimicking human biological fluids

A novel milk-derived peptide effectively inhibits Staphylococcus aureus: Interferes with cell wall synthesis, peptidoglycan biosynthesis disruption reaction mechanism, and its application in real milk system

CRISPR-associated protein 1 (Cas1) is a universally conserved essential metalloenzyme of the clustered regularly interspaced short palindromic repeat (CRISPR) immune system of prokaryotes (bacteria, archaea) that can cut and integrate a part of viral DNA to its host genome with the help of other proteins. The integrated DNA acts as a memory of viral infection, which can be transcribed to RNA and stop future infection by recognition (based on the RNA/DNA complementarity principle) followed by protein-mediated degradation of the viral DNA. It has been proposed that the presence of a single manganese (Mn2+) ion in a conserved divalent-metal-ion binding pocket (key residues: E190, H254, D265, D268) of Cas1 is crucial for its function. Cas1-mediated DNA degradation was proposed to be hindered by metal substitution, metal chelation, or mutation of the binding pocket residues. Cas1 is active toward dsDNA degradation with both Mn2+ and Mg2+. X-ray structures of Cas1 revealed an intricate atomic interaction network of the divalent-metal-ion binding pocket and opened up the possibility of modeling related metal ions (viz., Mg2+, Ca2+) in the binding pocket of wild-type (WT) and mutated Cas1 proteins for computational analysis, which includes (1) quantitative estimation of the energetics of the divalent-metal-ion preference and (2) exploring the structural and dynamical aspects of the protein in response to divalent-metal-ion substitution or amino acid mutation. Using the X-ray structure of the Cas1 protein from Pseudomonas aeruginosa as a template (PDB 3GOD), we performed (∼2.23 μs) classical molecular dynamics (MD) simulations to compare structural and dynamical differences between Mg2+- and Ca2+-bound binding pockets of wild-type (WT) and mutant (E190A, H254A, D265A, D268A) Cas1. Furthermore, reduced binding pocket models were generated from X-ray and molecular dynamics (MD) trajectories, and the resulting structures were subjected to quantum chemical calculations. Results suggest that Cas1 prefers Mg2+ binding relative to Ca2+ and the preference is the strongest for WT and the weakest for the D268A mutant. Quantum chemical calculations indicate that Mn2+ is the most preferred relative to both Mg2+ and Ca2+ in the wild-type and mutant Cas1. Substitution of Mg2+ by Ca2+ does not alter the interaction network between Cas1 and the divalent metal ion but increases the wetness of the binding pocket by introducing a single water molecule in the first coordination shell of the latter. The strength of metal-ion preference (Mg2+ versus Ca2+) seems to be dependent on the solvent accessibility of the divalent-metal-ion binding pocket, strongest for wild-type Cas1 (in which the metal-ion binding pocket is dry, which includes two water molecules) and the weakest for the D268A mutant (in which the metal-ion binding pocket is wet, which includes four water molecules).

An international team led by The Burnham Institute's Minoru Fukuda, Ph.D., has discovered that a human glycoproteininhibits Helicobacter pylori ("H. pylori"), the bacterium that causes stomach ulcers and is linked with 90% of stomach cancers. Published on August 13th in Science magazine, these results present a new way of looking at treating chronic inflammation associated with stomach ulcers, and introduces the possibility of preventing stomach cancer associated with H. pylori.Over fifty percent of the world's population is infected with H. pylori, yet only 2% are afflicted with stomach ulcers and only 1% with stomach cancer. A collaboration between The Burnham Institute and Japan's Shinsu University has discovered the defense mechanism that protects the stomach against H. pylori infection.H. pylori is found mostly in the stomach, where it thrives in the superficial mucin layer lining the stomach. The bacterium is rarely found in the deeper portion of the mucin layer, where the mucous cells produce a particular class of glycoproteins, called O-glycans, linked with the carbohydrate alpha 1,4-N-acetylglusosamine, cloned previously in Dr. Fukuda's laboratory. Because the alpha 1,4-linked N-acetylgucosamine is confined to the stomach's deeper mucosa lining, which also lacks H. pylori, the scientists investigated the possibility that it might play a role against infection by H. pylori.They isolated mucin from the upper and lower layers and found a key difference: surface-derived mucin actively supported H. pylori growth, while mucins from the second layer inhibited growth. H. pylori in the presence of alpha 1,4-linked N-acetylgucosamine lost its shape, became immobile, and eventually died. This cell-growth immobilizing effect is very similar to the effect of antibiotics, which dissolve or "lyse" the bacterium's cell wall.The researchers lysed H. pylori cells and set up a biochemical assay using mass spectrometry to analyze the bacterium's cell wall components. They discovered a cholesterol unique to H. pylori, cholesteryl-alpha-D-glucopyranoside, which is a major component of the bacterium's cell wall essential for its growth. Additional experiments confirmed that the O-glycans capped with alpha 1,4-linked N-acetylglucosamine blocked H. pylori's ability to synthesize the cholesterol."This is the first time that a glycoprotein has been shown to behave like an antibiotic," says Dr. Minoru Fukuda, who has devoted 20 years of his research career to the study of glycobiology and cancer. "This naturally-occurring cholesterol offers a very specific target for the design of safer drugs that could treat stomach ulcers and, long-term, prevent stomach cancer linked with H. pylori."Dr. Fukuda believes that it will be possible to breed cows and genetically engineer soy beans that produce milk bearing the inhibitory O-glycans capped with alpha 1,4-linked N-acetylglucosamine."This offers an inexpensive way to help people suffering in less developed countries," says Dr. Fukuda. "If we could use transgenic cows and plants to produce this milk, we could possibly eradicate H. pylori infection and eliminate stomach cancer."The Burnham Institute has applied for patents based on this work.Dr. Minoru Fukuda is a Professor in the Glycobiology Program of The Burnham Institute's Cancer Center. Other co-authors from The Burnham Institute include Dr. Michiko Fukuda, Professor, and Dr. Motohiro Kobayashi, postdoctoral fellow.This work was done in collaboration with Dr. Jun Nakayama, corresponding author, and Drs. Masatomo Kawakubo, Yuki Ito, Yukie Okimura, Kyoko Sakura, Susumu Kasama, and Tsutomu Katsuyama of Shinsu University, Japan.This research was supported by grants from the National Cancer Institute and the Department of Defense.

The coronavirus disease 2019 (COVID-19) virus has been spreading rapidly, and scientists are endeavouring to discover drugs for its efficacious treatment. Chloroquine phosphate, an old drug for treatment of malaria, has shown to have apparent efficacy and acceptable safety against COVID-19. As a part of Drug Discovery Hackathon-2020, in this study, the authors have tried making the derivatives of CQ and HCQ using MarvinSketch by ChemAxon. Molecular docking studies of these ligands were performed using Glide by Schrodinger, and ADME profiles were obtained by using QikProp. The obtained results after data analysis demonstrated that ligands HCQ_imidazoll, choloroquine_3c, HCQ_pyrrolC had good binding affinity and complied with all the ADME parameters. The molecular dynamic simulation of these ligands in complex with the 2019-nCoV RBD/ACE-2-B0AT1 complex PDB ID: 6M17 were carried out, and the parameters like RMSD, RMSF, and radius of gyration were observed to understand the fluctuations and protein-ligand interaction.

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Pheromone binding proteins (PBPs) are widely distributed in insect antennae, and play important roles in the perception of sex pheromones. However, the detail mechanism of interaction between PBPs and odorants remains in a black box. Here, a predicted 3D structure of PBP1 of the serious agricultural pest, Helicoverpa armigera (HarmPBP1) was constructed, and the key residues that contribute to binding with the major sex pheromone components of this pest, (Z)-11- hexadecenal (Z11-16:Ald) and (Z)-9- hexadecenal (Z9-16:Ald), were predicted by molecular docking. The results of molecular simulation suggest that hydrophobic interactions are the main linkage between HarmPBP1 and the two aldehydes, and four residues in the binding pocket (Phe12, Phe36, Trp37, and Phe119) may participate in binding with these two ligands. Then site-directed mutagenesis and fluorescence binding assays were performed, and significant decrease of the binding ability to both Z11-16:Ald and Z9-16:Ald was observed in three mutants of HarmPBP1 (F12A, W37A, and F119A). These results revealed that Phe12, Trp37, and Phe119 are the key residues of HarmPBP1 in binding with the Z11-16:Ald and Z9-16:Ald. This study provides new insights into the interactions between pheromone and PBP, and may serve as a foundation for better understanding of the pheromone recognition in moths.

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

The global pandemic crisis, COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed the lives of millions of people across the world. Development and testing of anti-SARS-CoV-2 drugs or vaccines, are not turned to be realistic in the timeframe needed to combat this pandemic. Thus, rigorous efforts are still ongoing for the drug repurposing as a clinical treatment strategy to control COVID-19. Here we report a comprehensive computational approach to identify the multi-targeted drug molecules against the SARS-CoV-2 proteins, which are crucially involved in the viral-host interaction, replication of the virus inside the host, disease progression and transmission of coronavirus infection. Virtual screening of 72 FDA approved potential antiviral drugs against the target proteins: Spike (S) glycoprotein, human angiotensin-converting enzyme 2 (hACE2), 3-chymotrypsin-like cysteine protease (3CLpro), Cathepsin L, Nucleocapsid protein, RNA-dependent RNA polymerase (RdRp) and nonstructural protein 6 (NSP6) resulted in the selection of seven drugs which preferentially binds to the target proteins. Further, the molecular interactions determined by MD simulation, free energy landscape and the binding free energy estimation, using MM-PBSA revealed that among 72 drug molecules, catechin (flavan-3-ol) can effectively bind to 3CLpro, Cathepsin L, RBD of S protein, NSP-6, and Nucleocapsid protein. It is more conveniently involved in key molecular interactions, showing binding free energy (ΔGbind) in the range of -5.09 kcal/mol (Cathepsin L) to -26.09 kcal/mol (NSP6). At the binding pocket, catechin is majorly stabilized by the hydrophobic interactions, displays ΔEvdW values -7.59 to -37.39 kcal/mol. Thus, the structural insights of better binding affinity and favourable molecular interaction of catechin towards multiple target proteins, signifies that catechin can be potentially explored as a multitargeted agent in the rational design of effective therapies against COVID-19.

A computational study on the molecular mechanisms of panduratin A as a potential inhibitor on SARS-CoV-2 protein targets

We present a new approach to more accurately and efficiently compute the absolute binding free energy for receptor-ligand complexes. Currently, the double decoupling method (DDM) and the potential of mean force method (PMF) are widely used to compute the absolute binding free energy of biomolecular complexes. DDM relies on alchemically decoupling the ligand from its environments, which can be computationally challenging for large ligands and charged ligands because of the large magnitude of the decoupling free energies involved. In contrast, the PMF method uses a physical pathway to directly transfer the ligand from solution to the receptor binding pocket and thus avoids some of the aforementioned problems in DDM. However, the PMF method has its own drawbacks: because of its reliance on a ligand binding/unbinding pathway that is free of steric obstructions from the receptor atoms, the method has difficulty treating ligands with buried atoms. To overcome the limitation in the standard PMF approach and enable buried ligands to be treated, here we develop a new method called AlchemPMF in which steric obstructions along the physical pathway for binding are alchemically removed. We have tested the new approach on two important drug targets involving charged ligands. One is HIV-1 integrase bound to an allosteric inhibitor; the other is the human telomeric DNA G-quadruplex in complex with a natural product protoberberine buried in the binding pocket. For both systems, the new approach leads to more reliable estimates of absolute binding free energies with smaller error bars and closer agreements with experiments compared with those obtained from the existing methods, demonstrating the effectiveness of the new method in overcoming the hysteresis often encountered in PMF binding free energy calculations of such systems. The new approach could also be used to improve the sampling of water equilibration and resolvation of the binding pocket as the ligand is extracted.

An accurate estimation of binding free energy of a ligand to receptor ΔG(bind) is one of the most important problems in drug design. The success of solution of this problem is expected to depend on force fields used for modeling a ligand-receptor complex. In this paper, we consider the impact of four main force fields, AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L, on the binding affinity of Oseltamivir carboxylate to the wild-type and Y252H, N294S, and H274Y mutants of glycoprotein neuraminidase from the pandemic A/H5N1 virus. Having used the molecular mechanic-Poisson-Boltzmann surface area method, we have shown that ΔG(bind), obtained by AMBER99SB, OPLS-AA/L, and CHARMM27, shows the high correlation with the available experimental data. They correctly capture the binding ranking Y252H → WT → N294S → H274Y observed in experiments (Collins, P. J. et al. Nature 2008, 453, 1258). In terms of absolute values of binding scores, results obtained by AMBER99SB are in the nearest range with experiments, while OPLS-AA/L, which is applied to study binding of Oseltamivir to the influenza virus for the first time, gives rather big negative values for ΔG(bind). GROMOS96 43a1 provides a lower correlation as it supports Oseltamivir to be more resistant to N294S than H274Y. Our study suggests that force fields have pronounced influence on theoretical estimations of binding free energy of a ligand to receptor. The effect of all-atom models on dynamics of the binding pocket as well as on the hydrogen-bond network between Oseltamivir and receptors is studied in detail. The hydrogen network, obtained by GROMOS, is weakest among four studied force fields.

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of wild type (WT) and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

Cyclooxygenase-2 (COX-2) is an enzyme involved in inflammation. The overexpression of COX-2 causes chronic inflammation, which can be prevented by COX-2 inhibitors. Generally, COX-2 inhibitors possess a carboxyl group and an aromatic ring in their molecular structure. These moieties are involved in the interaction with the active site of COX-2, thus playing a pivotal role in the inhibitory activity. Regarding the requisite molecular structure of COX-2 inhibitors, derivatives of dihydropyrimidinone (DHPM) are ideal candidates to be explored as COX-2 inhibitors, due to the ease of synthesis and their versatility to be transformed chemically. In this study, we prepared a novel small library consisting of 288 designed DHPM derivatives by varying the constituent components. The selection criteria of potential candidates for the COX-2 inhibitor of the data bank involve in silico studies via molecular docking investigations, prediction of ADMET and druglikeness, as well as molecular dynamics (MD) simulations. Molecular docking served as the initial step of selection, based on the comparison of grid score, docking pose, and interactions with those of lumiracoxib (LUR) as the original ligand of COX-2. The next criteria of selection were scores obtained from the ADMET and druglikeness by comparing the designed candidates with COX-2 inhibitors that were already marketed. Compound RDUE2 and SDT29 were the most potential candidates, which were further analyzed using the MD simulation. The results of the MD simulation indicated that RDUE2 and SDT29 interacted stably with amino acid residues on the active site of COX-2. The estimation of binding free energy indicated that SDT29 exhibited an inhibitory activity comparable to that of LUR, whereas RDUE2 showed a lower inhibitory activity than that of SDT29 and LUR.